Development of poly(lysine-g-(lactide-b-ethylene glycol)) terpolymers for nanoparticulate DNA packaging.

摘要

Non-viral vectors for gene therapy must overcome many extracellular and intracellular hurdles in order to achieve expression. Polylysine is one such polycation that has been studied extensively as a vehicle for transfection. It has been proven to spontaneously associate with DNA to form sub-micron particles; however, it has also been implicated as a cause of cytotoxicity and results in transient expression levels. In this work, a polylysine-based terpolymer of poly(lysine-g-(lactide-b-ethylene glycol)) has been developed that addresses some of the weaknesses of native polylysine transfection. One goal was to minimize the molecular weight of polylysine needed to completely condense DNA in order to reduce cytotoxicity, as well as to encourage intracellular unpackaging. The polylactide segment of the terpolymer was designed to help polylysine condense DNA and encapsulate it for controlled release. In addition, the hydrophilic polyethylene glycol chains served to improve colloidal stability of complexes and provided a spacer arm for conjugation of bioactive ligands. The composition of terpolymers was varied and characterized with respect to binding to DNA and resulting complex characteristics. Low molecular weight polymer components were utilized to encourage intracellular plasmid unpackaging and minimize toxicity. Grafting poly(lactide-b-ethylene glycol) onto polylysine (Mw = 836, 2926, 8800) decreased the amount of lysine residues needed for complete condensation by as much as 50% when compared to unmodified polylysine. Increasing the length of the polyactide segment decreased the minimum amount of polylysine necessary for condensation and was attributed to the increasing hydrophobic contribution. In addition, complexes made with terpolymers exhibited significantly better resistance against salt destabilization than those made with unmodified polylysine. Slow hydrolysis of polylactide enabled controlled, long term release of plasmid and the kinetics was dependent on the molecular weights of both the polylactide and polylysine segments, as well as the total amount of terpolymer used. In addition, one terpolymer composition was selected and used for transfection of osteoblast like MC3T3-E1 cells. The ability to use terpolymer composition to easily adjust complex characteristics can provide a means for optimization of the transfection system for sustained expression profiles of exogenous DNA.